1. Field of the Invention
The present invention relates to methods for producing secreted polypeptides. The present invention also relates to variant signal peptides or variant prepropeptides and nucleic acid constructs, vectors, and host cells comprising the variant signal peptide or variant prepropeptide coding sequences operably linked to polynucleotides encoding polypeptides.
2. Description of the Related Art
A signal peptide is an amino acid sequence linked in frame to the amino terminus of a polypeptide having biological activity and directs the encoded polypeptide into the cell's secretory pathway. A propeptide is an amino acid sequence positioned at the amino terminus of a polypeptide, wherein the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is linked in frame to the amino terminus of a polypeptide and the signal peptide region is linked in frame to the amino terminus of the propeptide region.
The secretion of a recombinant polypeptide of interest in a particular host cell may require the replacement of its native signal peptide with a new signal peptide that is compatible with the host cell of choice. In addition, a propeptide sequence of a polypeptide may also need to be replaced with a different propeptide sequence or deleted because the propeptide sequence is not processed or is processed improperly by the host cell to secrete an active polypeptide. In other cases, a signal peptide and/or propeptide may be absent.
It may be possible to improve the secretion of a polypeptide of interest by modifying the native signal peptide or native prepropeptide naturally associated with the polypeptide through mutation.
Belin et al., 2004, Journal of Molecular Biology 335: 437-453, describe mutations that improve the functional activity of the plasminogen activator inhibitor 2 (PAI-2) signal sequence. Tsuchiya et al., 2003, Nucleic Acids Research Supplement No. 3, pp. 262-262, describe mutation of a signal sequence for effective secretion of human lysozyme in yeast. Nothwehr and Gordon, 1990, The Journal of Biological Chemistry 265: 17202-17208, describe structural features in the amino terminal region of the human pre(Δpro)apolipoprotein A-II signal peptide that influences the site of its cleavage by signal peptidase. Ngsee and Smith, 1990, Gene 86: 251-255, describe changes in the bovine prolactin signal peptide required for efficient secretion in yeasts.
U.S. Pat. No. 5,766,912 discloses the cloning and sequence of a full-length wild-type lipase from Thermomyces lanuginosus containing a prepropeptide sequence. U.S. Pat. No. 5,869,438 discloses variants of a full-length wild-type lipase from Thermomyces lanuginosus. 
There is a need in the art for improved signal peptide and propeptide sequences for the secretion of active polypeptides in various host cells.
It is an object of the present invention to provide improved methods for producing a polypeptide in a fungal host cell using variant signal peptides or variant prepropeptides.